Deoxyribonuclease (DNase) is a phosphodiesterase capable of hydrolyzing polydeoxyribonucleic acid into individual 3′ or 5′-phosphate deoxynucleotides on hydrolysis of deoxyribonucleic acid (DNA). Based on their biochemical properties and enzymatic activities, DNase proteins have been classified as two types, DNase I and DNase II. DNase I proteins have a pH optimum near neutral and an obligatory requirement for divalent cations, and create 5′-phosphate deoxynucleotide products. DNase II has an acid pH optimum, can be activated by divalent cations, and produces 3′-phosphate deoxynucleotides on hydrolysis of DNA.
The nucleic acid encoding a human DNase I has been isolated, sequenced and the protein expressed in recombinant host cells, thereby enabling the production of human DNase I in commercially useful quantities. The DNA encoding other polypeptides having homology to human DNase I have also been identified.
Human DNase I has recently been used to reduce the viscoelasticity of pulmonary secretions (mucus) in such diseases as pneumonia and cystic fibrosis (CF), thereby aiding in the clearing of respiratory airways. One such pharmaceutical composition is described by U.S. Pat. No. 6,440,412, issued to Frenz, et al., for purified forms of DNase I in which the DNase is provided in a formulation for use in administering to patients suffering from pulmonary distress.
One such formulation is sold under the tradename Pulmozyme® (dornase alfa, Genentech, USA), in which the recombinant human deoxyribonuclease I (rhDNase) is provided to a patient in an inhaled solution that is sterile, clear, colorless, and contains a highly purified solution of rhDNase. The characteristics of the rhDNase were selected to attack pulmonary secretions of persons having such diseases are complex materials, which include mucus glycoproteins, mucopolysaccharides, proteases, actin and DNA. rhDNase I was found to be effective in reducing the viscoelasticity of pulmonary secretions by hydrolyzing, of degrading, high-molecular-weight DNA that is present in such secretions.
A hyperactive rhDNase is described by U.S. Pat. No. 6,391,607, issued to Lazarus, et al., for human DNase I hyperactive variants, in which amino acid sequence variants of human DNase I that have increased DNA-hydrolytic activity are disclosed. The patent disclosure describes nucleic acid sequences encoding such hyperactive variants, thereby enabling the production of these variants in quantities sufficient for clinical use. The invention also relates to pharmaceutical compositions and therapeutic uses of hyperactive variants of human DNase I.